Epibiotic microorganisms associated with ...

Soil Science and Plant Nutrition

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Epibiotic microorganisms associated with microcrustaceans in overlying water of Philippine paddy fields Misako Taniguchi , Wilbur Ventura , Koki Toyota & Makoto Kimura To cite this article: Misako Taniguchi , Wilbur Ventura , Koki Toyota & Makoto Kimura (1999) Epibiotic microorganisms associated with microcrustaceans in overlying water of Philippine paddy fields, Soil Science and Plant Nutrition, 45:3, 757-766, DOI: 10.1080/00380768.1999.10415841 To link to this article: http://dx.doi.org/10.1080/00380768.1999.10415841

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Date: 26 December 2016, At: 02:25

Short Paper Soil Sci. Plant Nu tr. , 45 (3), 757-766, 1999

757

Epibiotic Microorganisms Associated with Microcrustaceans in Overlying Water of Philippine Paddy Fields Misako Taniguchil, Wilbur Ventura*, Koki Toyota, and Makoto Kimura Laboratory of Soil Biology and Chemistry, School of Agricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan; and' International Rice Research Institute, P.D. Box 933, 1099 Manila, Philippines Received February 10, 1999; accepted in revised form May 31, 1999

Epibiotic microorganisms associated with micro crustaceans in the overlying water of Philippine paddy fields were examined by scanning electron microscopy, and their associations were compared with those observed in Japanese paddy fields (Soil Sci. Plant Nutr., 43, 633-641, 651-664, 1997). Various types of microorganisms, including bacteria, protozoa, and algae, colonized microcrustaceans and their kinds and colonizing patterns varied depending on the kinds of microcrustaceans. Common associations between epibionts and respective micro crustaceans were observed in the Philippines and in Japan: e.g., rodshaped bacteria on Cyclopidae and Cypridopsidaej no microorganisms on Simocephalus spp. Key Words: paddy field.

cladoceran, copepod, epihiotic microorganism, microcrustacean,

It is well known that various kinds of organisms, such as bacteria, protozoa, algae, and rotifers, colonize the surface of microcrustaceans living in the sea, lakes (Green 1974; Holland and Hergenrader 1981; Nagasawa 1989), and paddy fields (Taniguchi et al. 1997b). It was reported that the excreta of copepods in aquatic environments stimulated heterotrophic bacterial activity (Carman 1994), and that epibiotic bacteria accounted for 1-10% of the total bacterial number in the overlying water in a Japanese paddy field (Taniguchi et al. 1997a). Thus, the epibiotic bacteria on microcrustaceans may play significant roles in the functions and diversity of bacterial communities in the overlying water. In general, the kind of epibiotic microorganisms, their density and the site of colonization were reported to be specific for respective microcrustaceans (Taniguchi et al. 1997a, b). According to the studies conducted under both greenhouse and field conditions by these authors, bacteria with a very similar morphology, apparently belonging to a single type, colonized the surface of some kinds of microcrustaceans, e.g. Cypretta spp., throughout the flooding period in a Japanese paddy field, while no epibiotic microorganisms were observed on Simocephalus spp. In contrast, the composition of epibionts of Moina spp. varied during the flooding period. To determine whether such relationships between the epibiotic microorganisms and microcrus1 Present address: Genetic Stock Research Center, National Institute of Genetics, Mishima, 411-8510 Japan. To whom correspondence should be addressed.

758

M. TANIGUCHI et al.

taceans observed in Japanese paddy fields are ubiquitous, we studied the epibiotic microorganisms associated with microcrustaceans in Philippine paddy fields, which differ in location, climate, field management, etc. Materials and methods Experiment 1. Microcrustaceans (at least 100 individuals from each field) were collected with a plastic cup through a plankton net (mesh size 40,um) from various paddy fields located around Laguna Bay, Laguna State, Luzon Island, Philippines, just before the harvesting time in the 1996 dry season (April 27 to May 2, 1996). Experiment 2. Microcrustaceans were collected from the long-term biofertilizer experimental paddy fields at the International Rice Research Institute CIRRI), Philippines, 4 times during the 1996 dry season. A total of 0.5 L overlying water which was collected with a plastic cup from several areas in each plot was filtered through a plankton net (mesh size 40 ,urn). The experimental plots were subjected to four treatments as follows: control (no N fertilizer was applied), and plots with application of either urea, harvested Azolla spp. or harvested Sesbania spp. The plots were set up in 1985 and details on field management were reported elsewhere (Ventura and Watanabe 1993). Rice plants (IR 72) were transplanted on February 1, 1996 and harvested on May 20. Panicle initiation started on March 26 and flowering occurred on April 21. Enumeration of micro crustaceans. Microcrustaceans collected were fixed in sugar formalin (9.7% sugar; 4% formalin) and preserved at 4"C. Microcrustaceans were counted separately in terms of order, family or genus level under a magnification of X 10 to X 100, according to the methods of Mizuno (1987) and Mizuno and Takahashi (1991). Observation of epibiotic microorganisms on micro crustaceans with a scanning electron microscope (SEM). Microcrustaceans preserved were then fixed in glutaraldehyde (2% in 0.1 M phosphate buffer; pH 7.3) and dehydrated through a graded series of ethanol-isoamyl acetate (1: 1, v Iv) and finally stored in isoamyl acetate for critical drying using CO 2 (critical point drier; HITACHI HCP-l). Dried samples were mounted and sputtercoated with gold palladium (ion sputter; HITACHI E-1030) and observed with a scanning electron microscope (HIT ACHI S-4200K). Types of epibiotic microorganisms were identified from their size under SEM and the presence of a green color by microscopy. Results and discussion 1. Enumeration of micro crustaceans Experiment 1. Nine taxa of microcrustaceans were identified in paddy fields distributed around Laguna Bay (Table 1). All the 9 taxa were those commonly observed in Japanese paddy fields, especially Moina spp. and Cyclopidae are predominant microcrustaceans in Japanese paddy fields (Takahashi 1955; Kurasawa 1956; Takaku et al. 1979; Taira and Hogetsu 1987; Taniguchi et al. 1997a, b). Experiment 2. Composite data of the 4 plots are shown in Table 2. Ten taxa of microcrustaceans were identified (Table 2). Diaphanosoma spp., Macrothrix spp., Moina spp., Cyclopidae, and /lyocypris spp. were also observed in Experiment 1, while Cypridopsidae and Cypris spp. observed in Experiment 1 were absent in Experiment 2. Moina spp., Cyclopidae, and /lyocypris spp. were commonly observed in this study and our previous studies (Taniguchi et al. 1997a, b). The number and composition of micro crustaceans varied markedly among the 4 plots. The range (235 to 524 L -1) of the numbers observed was roughly comparable to that (24 to 1,870 L -1) in a Japanese paddy field (Taniguchi et al.

759

Epibionts of Microcrustaceans in Philippine Paddy Fields Table 1.

Microcrustaceans sampled from Philippine paddy fields in 1996.

Taxon Cladocerans Diaphanosoma spp. Macrothrix spinosa Moina spp. Simocephales spp. Copepods Cyc10pidae Ostracods Cypridopsidae Cypris spp. Ilyocypris spp. Others

Sampling site Luisiana IRRI* Paete, Siniloan, Batangas Bay Siniloan, IRRI, Liliw, Luisiana, Magdaena, Batangas, Tiaong Siniloan, Magdaena, Batangas Calauan, Paete, Siniloan, Batangas, Bay, Tiaong Liliw, Magdaena Magdaena

* Paddy fields at IRRI other than the long-term biofertilizer experimental paddy fields.

Table 2. Changes in numbers and composition of microcrustaceans in the overlying water of the long-term biofertilizer experimental paddy fields in IRRI during the growth period of rice. Date

February 19

March 8

April 22

April 29

14

32

77

84

423±246*

447±653

524±450

235± 142

Days after transplanting Total numbers (No. L -1) Composition (%) Cladocerans Ceriodaphnia cornuta Chydoridae Diaphanosoma spp. Macrothrix spp. Moina spp. Scapholeberis spp. Copepods Cyc10pidae Calanoida Harpacticoida Nauplius Ostracods Ilyocypris spp. Others

4.3*' 1.2 3.2 0 ll.! 2.3 8.2 0.6 0.2 68.8

(0-8.1)'" (0.6-3.3) (0-15.7) (0.6-16.9) (1.2-3.7)

54.6 1.7 0.1 0.8 1.0 0.5

(0-68.9) (0-8.6) (0-0.1) (0-43.8) (0-6.3) (0-0.6)

(3.3-12.3) (0-1.6) (0-0.8) (51.8-79.9)

10.5 0.8 0.3 28.5

(3.1-40.7) (0.4-12.5) (0-7.7) (12.5-46.3)

0.3 0.3 1.3 0 0.5 0

(0-1.4) (0-1.3) (0-2.9) (0-7.4)

38.2 (7.4-52.3) 1.5 (0-25.9) 0 57.6 (46.3-81.1)

0.4 (0-0.8) 0 0 0 0 0 0.6 (0-2.6) 7.9 (0.8-12.8) 0 89.8 (82.1-95.5)

0.2 (0-0.5) 0.9 (0-2.3) 0.1 (0-0.6) 0.3 (0-7.7) 0.2 (0-2.6) 0 0.8 (0-15.4) 0 'Mean among the 4 plots ± standard deviation. Mean composition (%) of the 4 plots .••• Figures parentheses show a range of composition (%) of the 4 plots.

*.

III

1997b). N auplii, copepods in the juvenile stage (thereby impossible to classify further), appeared to be predominant micro crustaceans throughout the flooding period. Cladocerans accounted for a significant proportion of the micro crustaceans in the early flooding period, while micro crustaceans observed consisted exclusively of copepods in the late period. Such observations was coincided with those made in a Japanese paddy field (Taniguchi et al. 1997b). Few ostracods were observed throughout the flooding period in this paddy field.

760

2.

M. TANIGUCHI et al.

SEM observation of micro crustaceans Experiment 1 Cladocerans Moina spp.: Moina spp. were observed in three paddy fields in Paete, Batangas, and

Siniloan. Rod-shaped bacteria with a similar shape (size 0.4 X I ,urn) colonized the exoskeleton of Moina spp. in Paete (Fig. la) (3 out of 3 individuals), while rods or filamentous bacteria in Batangas (Fig. I b) (2 out of 2), and no microorganisms were observed on the exoskeleton in Siniloan (Fig. lc) (8 out of 8). Diaphanosoma spp.: Rods colonized only the periphery of the oral region, and no microorganisms were observed in the other parts of the exoskeleton (4 out of 4). Macrothrix spinosa: Several types of rods colonized the exoskeleton (6 out of 8).

a

b

c

Fig. 1. Epibiotic microorganisms colonizing the surface of Moina spp. collected from paddy fields in the Philippines. a: Rod-shaped bacteria on exoskeleton of Moina spp. in Paete. b: Rods or filamentous bacteria on exoskeleton of Moina spp. in Batangas. c: No bacteria on exoskeleton of Moina spp. in Siniloan. Scale bars indicate I /-lm (a, b) and 5 /-lm (c).

Epibionts of Microcrustaceans in Philippine Paddy Fields

761

Simocephalus spp.: No microorganisms were observed on the surface of Simocephalus spp. (5 out of 5). Also no microorganisms were found on the surface of Simocephalus spp. in a Japanese paddy field (Taniguchi et al. 1997b).

Copepods Cyclopidae: In most of the Cyclopidae rods colonized the surface in Siniloan (5 out of 6), Laguna (8 out of 9), Liliw (2 out of 2), Magdaena (Fig. 2a) (2 out of 2), Tiaong (Fig. 2b) (2 out of 3), and at IRRI (Fig. 2c, d) (3 out of II) (a paddy field at IRRI other than the long-term biofertilizer experimental paddy fields). No algae were observed on the Cyclopidae collected from any of the sampling sites.

Ostracods Cypridopsidae: Protozoa and rods colonized the valves (Fig. 3a) (2 out of 2) in Siniloan and rods colonized the valves (4 out of 4) in Magdaena (Fig. 3b). These results were in agreement with those obtained in our previous study, which revealed that in Cypridopsis spp. rods and protozoa colonized the surface of the valves while some rods were attached to the valves on the end (Taniguchi et al. 1997b). Cypris spp.: Various microorganisms, including bacteria and algae, colonized the valves of Cypris spp. collected from Calauan (Fig. 4a) (11 out of II), Siniloan (Fig. 4b) (8 out of 8), and Laguna Bay (10 out of 10). Several types of rods colonized the valves of Cypris spp. collected from Paete (Fig. 4c) (IS out of IS) and Tiaong (Fig. 4d) (8 out of 8). Prosthecate bacteria colonized the valves of Cypris spp. collected from Batangas (Fig. 4e) (2 out of 2).

a

c

b

d

Fig. 2. Epibiotic microorganisms colonizing the surface of Cyclopidae collected from paddy fields in Magdaena (a), Tiaong (b), and IRRI (c, d), Philippines. Scale bars indicate 1 }lm.

762

M. TANIGUCHI et al.

a

b

Fig. 3. Epibiotic microorganisms colonizing the surface of Cypridopsidae collected from paddy fields in the Philippines. a: Protozoa and rods colonizing the valves of Cyclopidae. b: Rodshaped bacteria colonizing the valve of Cyclopidae on their end. Scale bars indicate 5 J.lm (a) and I J.lm (b).

Collectively, various kinds of microorganisms were known to colonize the valves of Cypris spp. These observations were consistent with those made in our previous experiment under laboratory conditions (Fig. 4f), while only a few types of microorganisms colonized the valves of Cypris spp. in a Japanese paddy field (Taniguchi et al. 1997b). Ilyocypris spp.: Rods colonized the valves of Ilyocypris spp. collected from Liliw (Fig. Sa) (3 out of 3) and Magdaena (lout of 1). In contrast, debris and organisms like algae or protozoa were frequently observed on the valves of /lyocypris spp. in Experiment 2 (Fig. 5b) and in a Japanese paddy field (Taniguchi et al. 1997b). Unidentified ostracods #1: Twisted long rods colonized the valves (5 out of 5).

Experiment 2 Epibiotic microorganisms on micro crustaceans in the respective 4 plots were first observed separately with SEM. No differences in the kind ofepibionts and their density were recognized by SEM observation among the 4 plots, although they changed according to the kind of microcrustaceans and the growth stage of the rice plant. The results obtained in the IRRI experimental fields are as follows.

Cladocerans Ceriodaphnia corn uta: Algae and rod-shaped bacteria colonized the exoskeleton on February 19 (Fig. 6a, b) (7 out of 15 individuals) and March 8 (8 out of 13), but few microorganisms were observed on the exoskeleton on April 22 (2 out of 2). Chydoridae: Rods were observed only in the periphery of the oral region on February 19 (2 out of 8) and April 22 (2 out of 2). In a Japanese paddy field, slightly-curved rods colonized the exoskeleton (Taniguchi et al. 1997b), although no microorganisms were


763

a

c

e

Fig. 4.

Epibiotic microorganisms colonizing the surface of Cypris spp. collected from paddy fields in the Philippines. Algae and bacteria colonizing the valves of Cypris spp. in Calauan (a) and Siniloan (b). Several types of rods colonizing the valves of Cypris spp. in Paete (c) and Tiaong (d). Prosthecate bacteria colonizing the valves of Cypris spp. in Batangas (e). Algae and bacteria colonizing the valves of Cypris spp. collected from a greenhouse study in Japan (t). Scale bars indicate I ,urn (d, e), 5,um (a, c), and lO,um (b, t).

observed on the exoskeleton except for the oral region in this study. Diaphanosoma spp.: Rods colonized only the periphery of the oral region on April 22 (2 out of 2). Macrothrix spp.: No microbial colonization was observed on March 8 (6 out of 6). Moina spp.: Algae and rod-shaped bacteria colonized the exoskeleton on February 19 (13 out of 36). Rods colonized the exoskeleton on March 8 (7 out of 8). Few rods were observed to be attached to the exoskeleton and they did not appeared to colonize the surface

764

M. TANIG UCHI et al.

Fig. 5. Epibiotic microorganisms col o nizing the surface of llyocyp ris spp. coll ected from paddy fields in the Phil ippi nes. a: Rod-shap ed bacter ia co lo nizing the va lves of lly ocypris spp. in Lil iw. b: A lgae or protozoa co lo nizing the valves of Ilyocypris spp . at IRR I. Sca le bars ind icate I p m (a ) an d 10 p m (b).

Fig. 6. Epibiotic microo rganisms co loni zing the surface of Ceriodaphnia cornuta collected from pa ddy fields in the Philippines. Alga e (a) and ro dshape d bac teria (b) co lo nizing the va lves of C. cornuta. Scale bar s indicate 5 pm (a) and I p m (b) .

o n April 22 (5 out of 5). These observatio ns agreed well with the results in our previ o us study , where in Mo ina spp. a lgae and rods co lo nized the surface in th e early flood in g peri od and the reafter algae and then rods di sappear ed in a J ap anese paddy field (T aniguchi et al. 1997b ). The disappear ance of algae in th e late floodin g peri od may be rela ted to th e light intensity in the ove rlyi ng water of paddy fields, whereas the reaso n for the di sappear ance of bacteria was un known. Co nseq uen tly, epib iot ic microorgan isms of M oina spp. appeared in succe ssion throu ghout th e floodin g perio d, which may acco unt for th e fact tha t several colonizi ng patterns of epi bio nts were ob served 011 M oina spp. in Exp eriment 1. Scaplzoleberis spp.: Algae and rod-sh aped bacteria co lo niz ed the exoskeleton on Febru ary 19 (2 o ut of 2). Copepods Cyclo pidae: Algae and rods colonized the exoskeleto n on Februar y 19 (8 o ut of 13) and March 8 (10 ou t of II). R od s col o nized the exos keleto n on April 22 (20 o ut of21) an d April 29 (6 o ut of 8). No epibi oti c alg ae o n Cycl op idae in Experiment I and in the lat e period of Ex perime nt 2 were observed . Th is finding migh t be related to the light intensity in the ove rlying water of the padd y fields which decreas es with th e growth of rice pl an ts, resulti ng

765


in the decrease of algal activity (Ichimura 1955; Kurasawa 1956). Calanoida: No microorganisms were observed on most individuals on April 22 and 29 (11 out of 15). Harpacticoida: Long rods colonized the ventral surface on March 8 (2 out of 2).

Ostracods Ilyocypris spp.: A few kinds of rods colonized the valves on February 19 (1 out of 1). Algae or protozoa colonized the valves on March 8 (Fig. 5b) (2 out of 3). Similar organisms in terms of morphology were observed on Ilyocypris spp. in a Japanese paddy field (Taniguchi et al. 1997b). No microorganisms were observed on the valves on April 22.

Conclusion The present study demonstrated that various types of microorganisms, including bacteria, protozoa, and algae, colonized microcrustaceans, and that their kinds and colonizing patterns varied depending on the kinds of microcrustaceans. The composition of epibiotic microorganisms changed during the flooding period for some microcrustaceans, where the light intensity in overlying water and applications of agrochemicals could be

Table 3.

Epibiotic microorganisms of microcrustaceans in the overlying water of Philippine paddy fields. Paddy fields around Laguna Bay, Philippine

Cladocerans Ceriodaphnia corn uta

Chydoridae

Diaphanosoma spp. Macrothrix spp. Moina spp.

Long-term biofertilizer experimental paddy fields at TRRI February 19 March 8 April 22 April 29

*

rods + + algae+ +

*

rods + + no no

rods+ + rods + + rods + + no

*

rods+ + algae+ + rods + + algae+ +

rods +

*

rods+ +

*

rods+ +

*

* * *

*

*

*

rods+ + algae+ + no no no short rods+ rods+ +

a few types of bacteria+

Scapholeberis spp.

*

Simocephalus spp. Copepods Cyc\opidae

no

*

*

*

*

rods+ +

rods+ + algae+ + no

rods+ algae+ +

long rods + + no

*

algae+ +

rods++ algae+ + a few types of bacteria+ no rods + + no

*

no

long rods+ +

*

*

rods+ + algae+ protozoa+ + rods+ + protozoa+

rods+ +

protozoa + + no

rods+ short rods+

no

*

*

*

*

Calanoida

Harpacticoida Ostracods [lyocypris spp.

Cypridopsidae

*, not observed; no, no microorganisms; +, present; + +, abundant.

long rods+ long rods+ + a few types of no bacteria +

766

M. TANIGUCHI et al.

considered to be responsible for the fate of epibionts, but the explicit mechanisms remain to be elucidated. Based on our previous study in Japanese paddy fields (Taniguchi et al. 1997a, b), common occurrence in both paddy fields in the Philippines and Japan was as follows: 1)· Epibiotic microorganisms of Moina spp. appeared in succession throughout the flooding period as follows: algae and rod-shaped bacteria were major epibionts in the early period, then algae disappeared and finally no microorganisms were observed on the surface. 2) Rod-shaped bacteria were the predominant epibionts on Cyclopidae. 3) A single type of rods colonized the valves of Cypridopsidae in two different ways: on the end and along the entire length of the cells. 4) Various kinds of microorganisms including bacteria and algae tended to colonize the valves of Cypris spp. 5) No microorganisms were observed on Simocephalus spp. These observations may be of ecological importance, since they were commonly recorded at two very different locations, Philippines and Japan. Further studies should include the characterization and identification of the epibiotic microorganisms and the ecological significance of such microorganism-microcrustacean associations. Acknowledgments. The authors thank Prof. Y. Takeoka, Nagoya University, for his technical assistance in SEM observation.

REFERENCES Carman, K.R. 1994: Stimulation of marine free-living and epibiotic bacterial activity by copepod excretions. FEMS Microbiol. Ecol., 14, 255-265 Green, J. 1974: Parasites and epibionts of Cladocera. Trans. Zool Soc. Lond., 32, 417-515 Holland, R.S. and Hergenrader, G.L. 1981: Bacterial epibionts of diaptomid copepods. Trans. Am. Microsc. Soc., 98, 56-65 Ichimura, S. 1955: Ecological studies on the plankton in paddy fields. I. Seasonal fluctuation in the standing crop and productivity of plankton. Jpn. J. Bot., 14, 269-279 Kurasawa, H. 1956: The weekly succession in the standing crop of plankton and zoobenthos in the paddy field (I). Shigen Kagaku Kenkyujo Iho, 45, 86-99 (in Japanese) Mizuno, T. 1987: Illustrations of the Freshwater Plankton of Japan, 353 pp., Hoikusha Press, Osaka (in Japanese) Mizuno, T. and Takahashi, E. (Ed.) 1991: An Illustrated Guide to Freshwater Zooplankton in Japan, 532 pp., Tokai University Press, Tokyo (in Japanese) Nagasawa, S. 1989: Bacterial epibionts of copepods. Sci Prog. Oxf, 73, 169-176 Taira, M. and Hogetsu, K. 1987: Species composition of phyto- and zoo-plankton communities in fertilized and non-fertilized paddy fields. Jpn. J. Limnol., 48, 77-83 (in Japanese) Takahashi, E. 1955: Ecological studies on freshwater living organisms in Shonai District, Yamagata Prefecture ( I). Yamagata Norin Gakkaihou, 11, 26-32 (in Japanese) Takaku, T., Takahashi, M., and Otsuki, A. 1979: Dispersion of an organophosphorus insecticide, fenitrothion, in paddy fields and its effects on the microorganisms. Jpn. J. Limnol., 40, 137-144 (in Japanese) Taniguchi, M., Toyota, K., and Kimura, M. 1997a: Epibiotic bacteria associated with microcrustaceans in the overlying water of paddy fields. Soil Sci. Plant Nutr., 43, 633-641 Taniguchi, M., Toyota, K., and Kimura, M. 1997b: Seasonal variation of microcrustaceans and microbial flora on their surface in the overlying water of a Japanese paddy field. Soil Sci. Plant Nutr., 43, 651-664 Ventura, W. and Watanabe, I. 1993: Green manure production of Azolla microphylla and Sesbania rostrata and their long-term effects on rice yields and soil fertility. Bioi. Fertil. Soils, 15, 241-248